The ability to amplify the quantity of nucleic acid, especially specific nucleic acid sequences, in a sample is an important aspect of many molecular biology techniques and assays. Polymerase chain reaction (PCR), U.S. Pat. Nos. 4,683,195 and 4,683,202 has been widely used to achieve amplification of specific nucleic acid sequences. In this method a mixture of nucleic acid sequences is mixed with two short oligodeoxynucleotide primers which specify the specific sequences are to be amplified.
Many of the previous methods are related to amplification of DNA. However, there have been increasing attempts to amplify target RNA molecules. The amplification of RNA is important in areas such as expression analysis and viral detection. One technique involved in amplification of RNA is called RT-PCR. In this technique RNA molecules are copied into complementary DNA (cDNA) sequences by the action of reverse transcriptase. The cDNA is then amplified by DNA polymerase in conjunction with appropriate primers.
A separate methodology has been described by Van Gelder et al. U.S. Pat. Nos. 5,545,522, 5,716,785 and 5,891,636. Here RNA target molecules are reverse transcribed into cDNA by reverse transcriptase in conjunction with a primer which also combines a promoter sequence for T7 RNA polymerase. After double stranded cDNA has been produced, T7 RNA polymerase is added and multiple copies of complementary RNA (cRNA) are produced by transcription.
The method described by Van Gelder et al requires cDNA synthesis and is multi-step, requiring reverse transcriptase, RNAse, polymerase and ligase and also requires a purification step in the middle of the protocol. These additional steps add to the complexity and also cost of the synthesis of cRNA.
Recently it has been demonstrated that DNA dependent RNA polymerases (RNA polymerases) can replicate short fragments of RNA by transcription if the RNA molecule to be transcribed is attached to a double stranded DNA promoter. After transcription initiation by the RNA polymerase on the double stranded DNA region, transcription proceeds across the RNA-DNA junction and through the RNA region with no observable loss of speed or processivity. Additionally, the template RNA being transcribed can be single stranded RNA, double stranded RNA, or a DNA:RNA heteroduplex. The only requirement for this process being that the RNA polymerase must initiate transcription on a double stranded DNA segment (Arnaud-Barbe, et al. Nucleic Acid Research 26 3550-3554 (1998)).
DNA ligases catalyze the joining of DNA strands to one another, while RNA ligases catalyze the joining of RNA strands to one another. It is a common misconception that DNA ligase is very inefficient at ligation of DNA to RNA strands. It has been demonstrated, however, that DNA ligase catalyzes the efficient joining of 3′-OH-terminated RNA to 5′-phosphate-terminated DNA on a DNA scaffold (Arnaud-Barbe, et al, 1998). DNA ligase is much less effective at joining 3′-OH-terminated DNA to 5′-phosphate-terminated RNA (much like the nick present during Okazaki strand maturation prior to RNA primer removal) and is extremely weak at phosphodiester formation between two RNA strands (Sekiguchi and Shuman. Biochem 36: 9073-9079 (1997)).
Nath and Hurwitz J B C 249 3680-3688 (1974) described the covalent ligation of the 3′-OH of polyA to the 5′-phosphate of polydA provided a polydT sequence was present to provide hybridisation using either E-coli DNA ligase or T4 DNA ligase. Similar observations were reported by Fareed et al. (J. Biol. Chem. 246 925 (1971)).